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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV150028 1/1 Homo sapiens Cell culture supernatant dUC
Filtration
Koch R 2015 33%

Study summary

Full title
All authors
Koch R, Aung T, Vogel D, Chapuy B, Wenzel D, Becker S, Sinzig U, Venkataramani V, von Mach T, Jacob R, Truemper L, Wulf GG
Journal
Clin Cancer Res
Abstract
PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with agg (show more...)PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. EXPERIMENTAL DESIGN: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. RESULTS: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. CONCLUSIONS: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 1-10. ©2015 AACR. (hide)
EV-METRIC
33% (66th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
DNF
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
130.7 (pelleting)
Protein markers
EV: CD63/ CD81/ GAPDH/ ADAM10/ Flotillin2/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
130.7
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH"
ELISA
Detected EV-associated proteins
"ADAM10/ Flotillin2/ GAPDH"
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
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